PROJECT SUMMARY (ABSTRACT) The endogenous circadian clock that synchronizes cellular physiology with the earth's light/dark cycle affects all aspects of physiology ? from molecular to cellular to behavioral. The circadian system has an integral role in promoting cellular resilience in the face of environmental or internal perturbation. Circadian disruption has been linked with cardiovascular disease, obesity, pulmonary disease, and neuropsychiatric disorders, highlighting the importance of fundamental knowledge of cellular clock mechanisms for clinical medicine. While the molecular basis for circadian timekeeping has been exquisitely defined, how the clock responds to the environment to maintain cellular homeostasis remains mostly unknown. We will test the hypothesis that the core circadian clock protein CLOCK is a stress sensor that transmits environmental information through structural elements in its intrinsically disordered region to both the circadian timekeeping mechanism and the stress-response machinery. We have discovered that the RNA chaperone DDX3X critically regulates the sensitivity of CLOCK to environmental stimuli such as temperature and has potent effects on both circadian timekeeping and cellular resilience. We will determine how CLOCK mechanistically integrates with the cellular stress machinery, including the heat shock response. Beyond its role as a stress sensor, we will investigate how the circadian system and CLOCK adaptively organize the biochemical state of the proteome, sculpting the circadian assembly of biological pathways involved in homeostasis. We will test these cellular data in animals by measuring how genetic and behavioral models of circadian disruption and sleep deprivation re-program the rhythmicity of protein assemblies. Finally, we will apply these discoveries to human peripheral blood samples in which we have, for the first time, identified rhythms of protein synthesis. This proposal is strengthened by its multi-scaled and interdisciplinary approaches that harness the expertise of the PI in chronobiology, cell biology, and biochemistry in collaboration with experts in human circadian biology (Klerman), proteomics and phosphoproteomics (Asara), live cell and super-resolution imaging (Chen), and imaging and assay development (Barrett). Our hypothesis is that the molecular mechanisms for circadian resilience and their failure during stress underlie a maladaptive feedback loop that connects integrated cellular stress responses with circadian misalignment. Determining the mechanisms by which the circadian clock supervises, senses, and responds to environmental stimuli is a crucial challenge for linking circadian disruption to disease mechanisms. My clinical background and experience as a pediatric neurologist trained in sleep medicine supports and informs my research interest in sleep and circadian disorders. We anticipate that the successful testing of this hypothesis will offer novel and pharmacologically actionable targets for mitigating the bivalent link between circadian misalignment and the many human disorders associated with it. ! !